Osteocytes are mechanosensors in bone with an ability to control bone resorption and formation through the expression of RANKL and sclerostin, respectively. However, the mechanisms by which osteocytes convert local mechanical cues to chemical signals across the lacunocanalicular system (LCS) and to the bone surface remain unclear. Ca2+ dynamics are a hallmark response of osteocytes to mechanical loading, but few studies have explored the role of Ca2+-dependent events in osteocyte mechanotransduction. Preliminary studies from our laboratory have uncovered novel Ca2+-dependent events that could contribute to bone adaptation. We discovered that mechanical loading induces Ca2+-dependent actomyosin contractions in osteocytes and that flow-induced Ca2+ oscillations facilitate the release of extracellular vesicles (EVs) containing RANKL, OPG and sclerostin. We therefore hypothesize that mechanically-induced Ca2+/actomyosin dynamics spatially and temporally regulate key proteins to direct bone formation and resorption responses to mechanical loading. To test this hypothesis, we have designed specific aims that leverage multi-scale approaches to evaluate the relative influence of Ca2+ oscillations and actomyosin contractility on osteocyte protein expression using a combination of loading regimens and antagonists. In particular, we focus on RANKL, OPG, and sclerostin, as mechanisms underlying the mechanical modulation of these proteins remain unknown. Specific Aim 1: Simultaneously monitor Ca2+ and actin strains in quasi-3D and 2D networks of osteocytes subjected to various fluid shear conditions and measure EV release and gene/protein expression of RANKL, OPG, and sclerostin in the presence or absence of inhibitors for Ca2+ and actomyosin contractile pathways. Specific Aim 2: Simultaneously monitor Ca2+ and actin strains in osteocytes in ex vivo mouse tibiae subjected to various mechanical loads and quantify the short-term spatial distribution of EVs in the 3D osteocyte LCS in the presence or absence of inhibitors for Ca2+ and actomyosin contractile pathways. Specific Aim 3: Examine spatial and temporal protein expression of RANKL, OPG, and sclerostin in osteocytes and their relationship to EV markers and cross-sectional strain and periosteal/endosteal bone formation/resorption dynamics in mouse tibiae in vivo subjected to various mechanical loading conditions and antagonists for Ca2+ and actomyosin contractile pathways. We expect to demonstrate that Ca2+-induced actomyosin contractions and facilitated vesicle release is an important mechanism in the translation of mechanical loading to early biochemical responses and later bone adaptation. A better understanding of osteocyte mechanotransduction may improve our ability to prevent bone degeneration in diseases such as osteoporosis where this function is impaired. Furthermore, understanding how proteins like RANKL, OPG, and sclerostin are expressed under various conditions can inform the use of therapeutics targeting their expression and help optimize treatment strategies.